Functional Genomic Analysis of a RUNX3 Polymorphism Associated with Ankylosing Spondylitis

Matteo Vecellio, Liye Chen, Carla J Cohen, Adrian Cortes, Yan Li, Sarah Bonham, Carlo Selmi, Matthew A Brown, Roman Fischer, Julian C Knight, B Paul Wordsworth
Arthritis & Rheumatology 2020 December 26

OBJECTIVES: To investigate the functional consequences of the single nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter associated with ankylosing spondylitis (AS).

METHODS: The effects of rs4648889 on allele-specific transcription factor (TF) binding were investigated by DNA pull-down and quantitative mass spectrometry (qMS) with validation by electrophoretic mobility gel shift assays (EMSA), Western blot (WB) analysis of the pulled-down eluates, and chromatin immuno-precipitation (ChIP)-qPCR. Further functional effects were tested by siRNA knockdown of IRF5 followed by qRT-PCR and ELISA to measure mRNA and protein levels of IFNγ.

RESULTS: Using nuclear extracts from primary human CD8+ T-cells assessed by qMS, relative TF binding to the AS-risk "A" allele of rs4648889 was increased (3.7-fold, p<0.03) for IKZF3 (aiolos) and components of the NUcleosome Remodeling Deacetylase (NuRD) complex, including Chromodomain-Helicase-DNA-binding protein (CHD) 4 (3.6-fold, p<0.05) and Retinoblastoma-Binding Protein (RBBP) 4 (4.1-fold, p<0.02). In contrast, interferon regulatory factor (IRF) 5 bound significantly less to the A allele (8.2-fold, p=0.003). Validation with WB, EMSA and ChIP-qPCR confirm differential allelic binding for IKZF3, CHD4, RBBP4 and IRF5. Silencing of IRF5 in CD8+ T-cells increased IFNγ mRNA (measured by RT-qPCR (p=0.03) and protein by (ELISA p=0.02).

CONCLUSIONS: The findings suggest that the association of rs4648889 with AS reflects allele-specific binding of this enhancer-like region to certain TFs, including IRF5, IKZF3 and members of the NuRD complex. IRF5 may have crucial influences on CD8+ lymphocyte function that could reveal new therapeutic targets in AS.

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